Detection of cell surface antigens, particularly growth factor receptors, expressed at elevated levels by tumor cells as compared with normal cells is of particular concern in the fields of immunodiagnosis and immunotherapy of human tumors. See, e.g., H. F. Sears, et al, Cancer Res., 45:5910 (1985); H. Koprowski et al, Som. Cell Mol. Genet., 11:297 (1985); and H. Koprowski et al Proc. Nat'l. Acad. Sci., USA, 81:216 (1984). Efficient methods of detection of growth factor receptors would allow the determination of the growth requirements of tumor cell types and thus enable the development of therapeutic methods for reducing the growth and limiting the spread of such tumor cells in patients having certain cancers.
Presently three methods are primarily used to determine if a tumor cell or tumor cell line is expressing or over-expressing a particular receptor for a specific growth factor. One current approach to testing receptor expression is based on the analysis by radioimmunoassay ("RIA") of growth factor binding to the cell surface using intact cells. Such an assay is described in R. N. Fabricant et al, Proc. Nat'l. Acad. Sci., USA, 74:565 (1977); G. E. Landreth et al, Proc. Nat'l. Acad. Sci., USA, 77:4751 (1980); and D. D. Vale et al, Methods Enzymol, 109:21 (1985). However, such RIAs are frequently performed at 0 C to inhibit internalization of growth factor molecules. These methods are not particularly sensitive, lacking qualitative accuracy and frequently providing false negative results. A number of recent studies have indicated that the uptake and nuclear accumulation of growth factors in assays performed at 0.degree. C. do not reflect the dynamic aspects of growth factor binding under physiological conditions. Moreover, some surface receptors may be synthesized at levels below the detection limits of the assay. However, even at low levels such receptors may effectively transport growth factors into the cell and thus encourage the growth and multiplication of that particular tumor cell. See, e.g., B. A. Yanker et al, Proc. Nat'l. Acad. Sci., USA, 76:1269 (1979); E. M. Rakowicz-Szulczynska et al, Biochem. Biophys. Res. Comm., 140:174 (1986); E. M. Rakowicz-Szulczynska et al, Proc. Nat'l. Acad. Sci., USA, 83:3728 (1986); L. K. Johnson et al, Nature, 287:340 (1980); U. Y. Yeh et al, Proc. Nat'l. Acad. Sci., USA, 84:2317 (1987); M. B. Omary et al, Science, 238:1578 (1987).
Additionally such methods detect only those receptors expressed on the cell surface, and cannot detect receptors which are synthesized intracellularly and not expressed on the cell surface. Certain tumor cells synthesize both a receptor and an appropriate growth factor. Expression of the receptor on the cell surface may be undetectable because of a constant down regulation of the receptor by interaction with the synthesized growth factor in the cytoplasm. For example, some melanoma and colorectal carcinoma cells express platelet derived growth factor ("PDGF"), which is critical for their growth, but assays for the PDGF receptor fail to detect receptor on the cell surface. M. Keating and L. T. Williams, Science 239:914-916 (1988), reported that this result was due to an autocrine stimulation of v-sis oncogene-transformed cells. This involves an intracytoplasmic interaction of v-sis product with PDGF receptor and consequently results in an undetectable level of PDGF receptor expression on the cell surface. For these reasons, the RIA detection of growth factor binding to intact cells is a less than desirable methodology.
Another commonly used method for determining the presence and identity of growth factor receptors on tumor cells involves the use of monoclonal antibodies developed to these receptors. Binding of monoclonal antibodies to the cell surface is usually analyzed by immunoperoxidase staining, such as described by C. Ernst et al, Amer. J. Pathol., 117:451 (1984), the mixed hemadsorption assay described by M. Herlyn et al, J. Immunol., 134:4226 (1985), or by RIA as described by U. Rodeck et al, Cancer Res., 47:3692 (1987). This method however is very expensive and requires for its performance a monoclonal antibody capable of identifying the cell surface receptor with the appropriate specificity and binding affinity. Without the prior provision of such a monoclonal antibody, this method cannot be used.
Additionally, this method, while very sensitive, also restricts the analyses to the cell surface receptors. Thus, a negative result obtained in this method also does not eliminate the possibility of the intracellular interaction of a synthesized receptor in the cytoplasm with simultaneously synthesized growth factor by the same cell.
Finally, a third method conventionally employed to detect expression of growth factor receptors is the Northern blot analysis, which detects specific receptor mRNA. While this method is very sensitive it also requires the availability of specific DNA probes derived only for those growth factor receptor genes that are cloned and sequenced. Thus in the absence of the cloned receptor gene, this method does not provide an appropriate tool to detect receptors on tumor cells.
Methods for detecting and determining the nutritional requirements of tumor cells for certain growth factors enable the analysis of which growth factors may be selectively used for therapeutic applications, as described in M. B. Sporn and A. B. Roberts, Nature, 332:217-219 (1988).
There exists, therefore, a need in the art for additional methods of detecting and quantitating expression of growth factor receptors by tumor cells.